Whole blood was assessed for the following markers: HLA-DR-FITC (BioLegend, San Diego, CA, USA), CD16-APC (BD), PD-1-APC-Cy7 (BioLegend, San Diego, CA, USA), CD3-AF700 (BD), CD11c-PE (BD), CD28-PE-Cy5 (BioLegend), NKG2a-PE-Cy7 (Beckman Coulter, Brea CA, USA), CD163-PE/Dazzle594 (BioLegend), CD4-BV421 (BD), CD14-BV510 (BioLegend), CD20-BV570 (BioLegend), CD8a-BV605 (BD), CD123-BV650 (BD), PD-L1-BV711 (BioLegend), CD95-BV785 (BioLegend), and Ki-67-PerCP-Cy5.5 (BD). Briefly, 100 μL of whole blood was incubated with 100 μL of the marker panel (excluding Ki-67) and incubated for 20 min. The red blood cells were lysed with 1 mL of BD FACSLyse (BD) for 10 min. The cells were washed and fixed for 30 min using BD Cytofix/Cytoperm (BD) and incubated with Ki-67 antibody for 30 min at 4 °C, followed by a final wash in 1× BD Permwash (BD). Data acquisition and analysis was performed with FlowJo version 10.2 (BD). During the acquisition and analysis, species-specific patterns of cell phenotypes were taken into consideration [25 (link)]. After filtering for single cell events and removing granulocytes, immune cell populations were assessed according to the features listed in Table 1 .
Pd 1 apc cy7
Manufactured by BioLegend
Sourced in United States
PD-1-APC-Cy7 is a fluorescently-labeled antibody that binds to the programmed cell death protein 1 (PD-1). It can be used for the detection and analysis of PD-1 expression on cells by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 bv510, by BioLegend (2 mentions)
Cd4 bv421, by BioLegend (2 mentions)
Cd4 fitc, by BioLegend (2 mentions)
Ifn γ pe cy7, by BioLegend (2 mentions)
Anti mouse cd16 32, by Thermo Fisher Scientific (2 mentions)
Cd62l apc cy7, by BioLegend (2 mentions)
Perm wash, by BD (1 mentions)
Facs lyse, by BD (1 mentions)
Cd16 apc, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Cd11c pe, by BD (1 mentions)
Cd14 bv510, by BioLegend (1 mentions)
Cd28 pe cy5, by BioLegend (1 mentions)
Cd4 bv421, by BD (1 mentions)
Cd3 af700, by BD (1 mentions)
Hla dr fitc, by BioLegend (1 mentions)
Nkg2a pe cy7, by Beckman Coulter (1 mentions)
Cd163 pe dazzle594, by BioLegend (1 mentions)
Cd20 bv570, by BioLegend (1 mentions)
Pd l1 bv711, by BioLegend (1 mentions)
9 protocols using pd 1 apc cy7
1
Multiparametric Flow Cytometry of Immune Cells
2
Profiling T Cell Subsets and Tfh
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To determine the percentage of different T cell subsets, at least 1 × 106 PBMCs were suspended in isoflow sheath fluid (Beckman Coulter) and stained in a DuraClone IM T cell tube (Beckman Coulter, Miami, FL) according to the manufacturer's protocol. The T cell tube contained anti-CD45, CD3, CD8, CD4, CD45RA, and CCR7. Samples were measured by use of the Navios flow-cytometer (Beckman Coulter).
To determine the Tfh, at least 1 × 106 PBMC were washed by Fascflow (BD Biosciences, New Jersey, US) and stained with CD3 BV510 (Biolegend, California, US), CD4 BV421 (Biolegend, California, US), CXCR5 Alexa Fluor 647 (BD Biosciences, New Jersey, US), and PD1 APC-Cy7 (Biolegend, California, US) for 30 min at room temperature in the dark.
To determine the Tfh, at least 1 × 106 PBMC were washed by Fascflow (BD Biosciences, New Jersey, US) and stained with CD3 BV510 (Biolegend, California, US), CD4 BV421 (Biolegend, California, US), CXCR5 Alexa Fluor 647 (BD Biosciences, New Jersey, US), and PD1 APC-Cy7 (Biolegend, California, US) for 30 min at room temperature in the dark.
3
Detecting IL-21 Production in PBMC Subsets
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Patient's PBMC were stimulated for 3 days at 37°C, 5% CO2 and 95% humidity with CSFE-labeled, irradiated (40 Gy) donor PBMCs, depleted for CD3+ T cells, in culture medium. At the end of day 2 monensin and brefeldin A (GolgiStop and GolgiPlug, BD Biosciences) were added for 16 h in a concentration of 1:1,500 and 1:1,000, respectively, to allow the measurement of intracellularly accumulated cytokines in PBMCs. Thereafter, intracellular IL-21 was measured, and surface marker staining was used to investigate which subsets produced these cytokines. Monoclonal antibodies used for surface marker staining and intracellular cytokine staining were CD3 BV510 (BioLegend), CD4 BV421 (BioLegend), CD8 PerCP (BD Biosciences), CXCR5 APC (BD Biosciences), PD-1 APC-Cy7 (BioLegend), and IL-21 PE (Biolegend).
4
Multiparametric Characterization of Immune Cells from Cryopreserved Samples
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Cryopreserved PBMC/cord BMC (CBMC) were thawed, counted, and processed immediately for phenotypic assessment using two staining panels. Panel A consisted of surface staining with Zombie yellow (viability), CD25 FITC (BioLegend), Lag3 PE (Invitrogen), CTLA4 PE-CF594 (BD Biosciences), CD4 PerCP-Cy5.5 (BD Biosciences), CD3 Ax700 (BD Biosciences) followed by fixation and permeabilization using the eBioscience Foxp3 / Transcription Factor Staining Buffer Set (eBioscience). Intracellular staining was then performed with FoxP3 Ax647 (BD Biosciences), Granzyme B APC-fire750 (BioLegend), IL-10 BV421 (BioLegend) and TGFβ PE-Cy7 (BioLegend). Panel B consisted of surface staining with Zombie yellow (viability), CD4 FITC (BioLegend), CD3 PE-CF594 (BD Biosciences), GITR PerCP-Cy5.5 (BioLegend), TNFR2 PE-Cy7 (BioLegend), CD39 Ax700 (R&D Systems), PD1 APC-Cy7 (BioLegend) and TIGIT BV421 (BioLegend). Intracellular staining consisted of FoxP3 Ax647 (BD Biosciences) and IL-35 PE (BioLegend). Analysis was performed using the Galios instrument (Beckman Coulter).
5
Profiling Immune Checkpoint Markers in OE33 Cells
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OE33 cells were trypsinised and stained with zombie aqua viability (Biolegend, United States) dye. Antibodies used for OAC cell lines included: PD-L1-FITC, PD-L2-PE, TIGIT-PE/Cy7, PD-1-APC/Cy7 (Biolegend, United States), OE33P and OE33R cells were fixed with 1% paraformaldehyde solution and acquired using BD FACs CANTO II (BD Biosciences) using Diva software and analysed using FlowJo v10 software (TreeStar Inc.). Tumour tissue biopsies were stained with zombie aqua viability dye (Biolegend, United States) as per manufacturer’s recommendations.
6
Multicolor Flow Cytometry of PBMC
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We isolated peripheral blood mononuclear cells (PBMC) from peripheral blood by Ficoll gradient and we stored them in liquid nitrogen for batched analysis. For multicolor flow cytometry analyses, we used the following monoclonal antibodies: from Becton Dickinson (BD, Franklin Lakes, NJ), CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CD71-PE, IgD-PerCP-Cy5.5, CD25-APC-Cy7, CD138-BV421, CCR4-PE, CD27-PE, CD95-BV421, CD28-BV421, TIM3-BV421, CCR6-BV421, CCR7-AF700, CXCR3-PE, IL-2-PE, IL-17-BV786, and IFN-γ-PE-Cy7; from Biolegend (San Diego, CA), CD19-BV510, CD56-FITC, CD27-APC, CD127-FITC, CD57-PerCp-Cy5.5, PD-1-APC-Cy7, CXCR5-FITC, LAG3-APC, IL-10-PerCP-Cy5.5, and TNF-α-FITC. From eBioscience (San Diego, CA), CD4-PE-Cy7, CD21-PE, and CD24-APC-Cy7 were used. From Miltenyi Biotec (Auburn, CA), CD25-APC and anti-KLRG1-PE were used. From Beckman Coulter (Indianapolis, IN), CD38-PE-Cy7 was used.
We performed intracellular staining for IL-2, IL-4, IL-17, IFN-g, and TNF-a together with extracellular markers for CD4+, CD8+, and CD19+. Cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.
Data were acquired (> 1 × 106 events) on a 3-laser FACSLyric flow cytometer (BD Biosciences) and analyzed with FlowJo® software.
We performed intracellular staining for IL-2, IL-4, IL-17, IFN-g, and TNF-a together with extracellular markers for CD4+, CD8+, and CD19+. Cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.
Data were acquired (> 1 × 106 events) on a 3-laser FACSLyric flow cytometer (BD Biosciences) and analyzed with FlowJo® software.
7
Multiparametric Flow Cytometry Analysis
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Cells were initially blocked with anti-mouse CD16/32 (eBioscience) then stained with fluorochrome-conjugated antibodies following our previously published procedures (55 (link)). Zombie Aqua (BioLegend) staining was performed to exclude dead cells. For quantification of B cells in total splenocytes, the following anti-mouse antibodies were used: CD19-Pacific blue, CD27-PE, CD138-APC-Cy7, CD44-PerCP-Cy5.5, IgD-PE-Cy7, GL7-AF647. For splenic T cells, CD3-APC, CD4-FITC, CD8-PE, CD44-PerCP-Cy5.5, CD62L-APC-Cy7, CD69-Pacific blue, CXCR5-PerCP-Cy5.5, and PD-1-APC-Cy7 (BioLegend) were used. For myeloid cell analysis, the following anti-mouse antibodies were used: CD11b-PE, CD11c-PerCP-Cy5, F4/80-PE-Cy7 (BioLegend), Gr1-V540 (BD Bioscience). Analysis was performed with a BD FACSAria II flow cytometer (BD Biosciences). Flow cytometry data were analyzed with FlowJo.
8
Multiparameter Flow Cytometry Immunophenotyping
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The following fluorescence-labeled monoclonal antibodies (mAb) were used: CD3-FITC or PE/Cy5 (clone UCHT1), CD11c-FITC (3.9), CD16-APC/Cy7 (3G8), CD30-PE/Cy7 (BY88), CD33-PE, BV605 or PE/Cy5 (P67.6), CD56-PE/Dazzle™594 (N901), CD57-FITC (HCD57), CD62L-PE/Cy7 (DREG56), CD107-BV510 (H4A3), CD117-BV421 (104D2), KLRG1-APC/Fire 750 (SA231A2), PD1-APC/Cy7 (EH12-2H7), NKG2D-PE (1D11), NKp46-BV510 (9E2), NKp44-APC (P44-8), NKp30-BV785 (P30-15), Granzyme B-Pacific Blue (GB11), IFNγ PE/Cy7 (B27), Perforin-PE (dG9), TNFα PE/Dazzle™ 594 (all from Biolegend, CA, USA), CD3 PerCP-Vio700 (REA613), CD14-PerCP-Vio700 (REA599), CD33-APC, CD56 (REA196), anti-biotin-VioBright515 and 7-AAD (all Miltenyi Biotec), NKG2C-AF700 (134591) from R&D systems (MN, USA), and CD158b1,b2,j-PE/Cy5 (GL183), NKG2A-APC (Z199) purchased from Beckman Coulter (CA, USA). Flow cytometric analyses were performed on a CytoFLEX (Beckman Coulter) or MACSQuant 10 Analyzer (Miltenyi Biotec). Data analysis was performed on Kaluza 2.1.1 software.
9
Multi-parameter Flow Cytometry of Immune Cells
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Spleen and mesenteric lymph node (MLN) were collected and mashed in 70-µm cell strainers with C10 media (RPMI-1640 containing L-glutamine, 10% fetal bovine serum, 100 U/ml penicillin-streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 1% 100X MEM non-essential amino acids, 55 µM 2-mercaptoethanol, all from Life Technologies). For splenocytes, red blood cells were lysed with RBC lysis buffer (eBioscience). For FACS staining, cells were blocked by anti-mouse CD16/32 (eBioscience), stained with fluorochrome-conjugated antibodies, and analyzed with BD FACSAria Fusion flow cytometer (BD Biosciences). Anti-mouse antibodies used in this study include: CD3-FITC and B220-FITC (eBioscience), CD4-PerCP/Cy5.5, CD8-PE/Dazzle, PD-1-APC/Cy7, CXCR5-BV605, CD25-BV421, FOXP3-PE, CD44-APC, CD62L-APC/Cy7, CD138-PerCP/Cy5.5, CD19-BV421, CD273-PE/Dazzle, CD38-PE/Cy7 and GL7-APC (Biolegend). For intracellular staining, Foxp3/Transcription Factor Staining Kit was used to fix the cells (Biolegend). For intracellular staining of IL-10 producing cells, splenocytes or MLN cells were pre-stimulated for 4 hours with 500X Cell Stimulation Cocktail (Invitrogen) plus protein transport inhibitor (eBioscience) containing phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A and monensin; then stained with CD19-AF700, CD138-BV711, Foxp3-AF647 and IL-10-PE. FlowJo was used to analyze data.
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